The present invention provides novel prokinetic agents with superior pharmacological and pharmacokinetic properties for the treatment of gastrointestinal motility disorders. The invention relates to the fields of chemistry, medicinal chemistry, medicine, molecular biology, and pharmacology.
Gastrointestinal (xe2x80x9cGIxe2x80x9d) motility regulates the orderly movement of ingested material through the gut to insure adequate absorption of nutrients, electrolytes and fluids. Appropriate transit through the esophagus, stomach, small intestine and colon depends on regional control of intraluminal pressure and several sphincters that regulate forward movement and prevent back-flow of GI contents. The normal GI motility pattern may be impaired by a variety of circumstances including disease and surgery.
Disorders of gastrointestinal motility include, for example, gastroparesis and gastroesophageal reflux disease (xe2x80x9cGERDxe2x80x9d). Gastroparesis is the delayed emptying of stomach contents. Symptoms of gastroparesis include stomach upset, heartburn, nausea, and vomiting. Acute gastroparesis may be caused by, for example, drugs (e.g., opiates), viral enteritis, and hyperglycemia, and is usually managed by treating the underlying disease rather than the motility disorder. The most common causes of chronic gastroparesis are associated with long standing diabetes or idiopathic pseudo-obstruction, often with so-called xe2x80x9cnon-ulcerxe2x80x9d or xe2x80x9cfunctionalxe2x80x9d dyspepsia.
GERD refers to the varied clinical manifestations of reflux of stomach and duodenal contents into the esophagus. The most common symptoms are heartburn and dysphasia; blood loss may also occur from esophageal erosion. GERD may be associated with low tone and inappropriate relaxation of the lower esophageal sphincter and occurs with gastroparesis in about 40% of cases. In most cases, GERD appears to be treatable with agents that reduce the release of acidic irritant by the stomach (e.g., Prilosec) or agents that increase the tone of the lower esophageal sphincter (e.g., cisapride). Other examples of disorders whose symptoms include impaired gastrointestinal motility are anorexia, gall bladder stasis, postoperative paralytic ileus, scleroderma, intestinal pseudoobstruction, gastritis, emesis, and chronic constipation (colonic inertia).
These GI disorders are generally treated with prokinetic agents that enhance propulsive motility. Motilides are macrolide compounds such as erythromycin and its derivatives that are agonists of the motilin receptor. Evidence of the potential clinical utility of motilides includes their ability to induce phase III of Migrating Motor Complexes (xe2x80x9cMMCxe2x80x9d). MMC refers to the four phases (I-IV) of electrical activity displayed by the stomach and small intestine in the fasting state. Muscular contraction occurs in phases III and IV which coincide with a peristaltic wave that propels enteric contents distally during fasting. Other clinically relevant effects include: increase in esophageal peristalsis and LES pressure in normal volunteers and patients with GERD; acceleration of gastric emptying in patients with gastric paresis; and stimulation of gallbladder contractions in normal volunteers, patients after gallstone removal, and diabetics with autonomic neuropathy.
The discovery of motilides was serendipitous. Since the 1950""s, erythromycin A 1 has been known to cause GI side effects such as nausea, vomiting, and abdominal discomfort. These effects are now largely explained by the motilin agonist activity of erythromycin A and an acid catalyzed degradation production, 8,9-anhydro-6,9-hemiacetal 2, which is also known as the enol ether form. 
As illustrated by Scheme A, erythromycin A 1 undergoes an acid catalyzed rearrangement in the stomach to form the enol ether 2 which is then further degraded into the spiroketal 3. Both erythromycin A and the enol ether are motilin agonists but the spiroketal is not. Because the enol ether is approximately ten fold more potent as a motilin agonist than erythromycin A and does not also possess antimicrobial activity, the potential clinical uses of enol ether derivatives as prokinetic agents are being investigated.
Enol ether erythromycin derivatives under clinical investigation include EM-523 (4); EM-574 (5); LY267,108 (6); GM-611 (7); and ABT-229 (8) whose structures are shown below. See U.S. Pat. Nos. 5,578,579; 5,658,888; 5,922,849; 6,077,943; and 6,084,079 which are all incorporated herein by reference. 
Other motilides of potential interest include lactam enol ethers and lactam epoxide derivatives. See also U.S. Pat. Nos. 5,712,253; 5,523,401; 5,523,418; 5,538,961; 5,554,605 which are incorporated herein by reference.
In general, these and other previously disclosed macrolides are synthetically accessible compounds that are derived from erythromycin A or B. Because nature has not optimized the erythromycin structure for its prokinetic activity, it is likely that the potency of motilide agonists could be greatly enhanced. Compounds resulting from such efforts could be of significant benefit in the treatment of wide variety of diseases and conditions. The present invention provides such compounds.
The present invention provides novel macrolide compounds with superior pharmacological and pharmacokinetic properties for the treatment of gastrointestinal motility disorders. In one embodiment, the present invention provides compounds of the formulas 
wherein:
R is hydrogen or hydroxyl;
R1 is selected from the group consisting of hydrogen, hydroxyl, halide, NH2, OR9, 
where R9 is C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, aryl or heteroaryl and R10 and R11 are each independently hydrogen, C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, or aryl;
R2 and R3 are each independently selected from the group consisting of hydrogen, C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, aryl, alkylaryl, alkenylaryl, alkynylaryl or R2 and R3 together form a cycloalkyl or a cycloaryl moiety;
R4 is hydrogen or methyl;
R5 is hydrogen, hydroxyl, oxo, or together with R6 and the carbons to which they are attached form a cyclic carbonate;
R6 is hydrogen, hydroxyl, OR12 where R12 is C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, or together with R5 and the carbons to which they are attached form a cyclic carbonate;
R7 is methyl, C3-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, alkylaryl, alkenylaryl, alkynylaryl, amidoalkylaryl, amidoalkenylaryl, or amidoalkynylaryl;
R8 is C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, alkylaryl, alkenylaryl, alkynylaryl, amidoalkylaryl, amidoalkenylaryl, or amidoalkynylaryl; and,
x is a single or a double bond. These and other embodiments, modes, and aspects of the invention are described in more detail in the following description, the examples, and claims set forth below.
The present invention provides novel macrolide compounds with superior pharmacological and pharmacokinetic properties for the treatment of gastrointestinal disorders where enhanced GI motiliy is indicated or desired. The compounds of the present invention typically are derived from xe2x80x9cunnaturalxe2x80x9d erythromycins and generally differ from naturally occurring erythromycins A, B, C, and D by having a non-ethyl group (e.g., a group that is not xe2x80x94CH2CH3) or a substituted ethyl at C-13 and/or by having a hydrogen instead of the methyl group at C-6 (C-6 desmethyl compounds).
Definitions
Many of the inventive compounds contain one or more chiral centers. All of the stereoisomers are included within the scope of the invention, as pure compounds as well as mixtures of stereoisomers. Similarly, all geometric isomers are also included within the scope of the invention. Where the compounds according to this invention have at least one chiral center, they may accordingly exist as enantiomers. Where the compounds possess two or more chiral centers, they may additionally exist as diastereomers. It is to be understood that all such isomers and mixtures thereof are encompassed within the scope of the present invention. Furthermore, some of the crystalline forms for the compounds may exist as polymorphs and as such are intended to be included in the present invention. In addition, some of the compounds may form solvates with water (i.e., hydrates) or common organic solvents, and such solvates are also intended to be encompassed within the scope of this invention.
For use in medicine, the salts of the compounds of this invention refer to non-toxic xe2x80x9cpharmaceutically acceptable salts.xe2x80x9d Other salts may, however, be useful in the preparation of compounds according to this invention or of their pharmaceutically acceptable salts. Suitable pharmaceutically acceptable salts of the compounds include acid addition salts which may, for example, be formed by mixing a solution of the compound with a solution of a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, citric acid, tartaric acid, carbonic acid or phosphoric acid. Furthermore, where the compounds of the invention carry an acidic moiety, suitable pharmaceutically acceptable salts thereof may include alkali metal salts, e.g., sodium or potassium salts; alkaline earth metal salts, e.g., calcium or magnesium salts; and salts formed with suitable organic ligands, e.g., quaternary ammonium salts. Thus, representative pharmaceutically acceptable salts include the following: acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, calcium edetate, camsylate, carbonate, chloride, clavulanate, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, flimarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate, N-methylglucamine ammonium salt, oleate, pamoate (embonate), palmitate, pantothenate, phosphate/diphosphate, polygalacturonate, salicylate, stearate, sulfate, subacetate, succinate, tarnate, tartrate, teoclate, tosylate, triethiodide and valerate.
The present invention includes within its scope prodrugs of the compounds of this invention. In general, such prodrugs will be functional derivatives of the compounds that are readily convertible in vivo into the required compound. Thus, in the methods of treatment of the present invention, the term xe2x80x9cadministeringxe2x80x9d shall encompass the treatment of the various disorders described with the compound specifically disclosed or with a compound which may not be specifically disclosed, but which converts to the specified compound in vivo after administration to the patient. Conventional procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in xe2x80x9cDesign of Prodrugsxe2x80x9d, ed. H. Bundgaard, Elsevier, 1985.
Listed below are definitions of various terms used to describe this invention. These definitions apply to the terms as they are used throughout this specification, unless otherwise limited in specific instances, either individually or as part of a larger group.
The term xe2x80x9calkylxe2x80x9d refers to an optionally substituted straight, branched or cyclic hydrocarbons. xe2x80x9cAlkenylxe2x80x9d refers to an optionally substituted straight, branched, or cyclic chain hydrocarbon with at least one carbon-carbon double bond. xe2x80x9cAlkynylxe2x80x9d refers to an optionally substituted straight, branched, or cyclic hydrocarbon with at least one carbon-carbon triple bound. Substituted alkyl, substituted alkenyl, or substituted alkynyl refer to the respective alkyl, alkenyl or alkynyl group substituted by one or more substituents. Illustrative examples of substituents include but are not limited to alkyl alkenyl, alkynyl, aryl, halo; trifluoromethyl; trifluoromethoxy; hydroxy; alkoxy; cycloalkoxy; heterocyclooxy; oxo (xe2x95x90O); alkanoyl (xe2x80x94C(xe2x95x90O)-alkyl); aryloxy; alkanoyloxy; amino; alkylamino; arylamino; aralkylamino; cycloalkylamino; heterocycloamino; disubstituted amines in which the two amino substituents are selected from alkyl, aryl, or aralkyl; alkanoylamino; aroylamino; aralkanoylamino; substituted alkanoylamino; substituted arylamino; substituted aralkanoylamino; thiol; alkylthio; arylthio; aralkylthio; cycloalkylthio; heterocyclothio; alkylthiono; arylthiono; aralkylthiono; alkylsulfonyl; arylsulfonyl; aralkylsulfonyl; sulfonamido (e.g., SO2NH2); substituted sulfonamido; nitro; cyano; carboxy; carbamyl (e.g., CONH2); substituted carbamyl (e.g., xe2x80x94C(xe2x95x90O)NRxe2x80x2Rxe2x80x3 where Rxe2x80x2 and Rxe2x80x3 are each independently hydrogen, alkyl, aryl, aralkyl and the like); alkoxycarbonyl, aryl, guanidino, and heterocyclo such as indoyl, imidazolyl, furyl, thienyl, thiazolyl, pyrrolidyl, pyridyl, pyrimidyl and the like. Where applicable, the substituent may be further substituted such as with halogen, alkyl, alkoxy, aryl, or aralkyl and the like.
The term xe2x80x9carylxe2x80x9d refers to an optionally substituted aromatic ring having 6 to 12 carbon atoms and may include one or more heteroatoms such as N, S and O. Illustrative examples of aryl include but are not limited to biphenyl, furyl, imidazolyl, indolyl, isoquinolyl, naphthyl, oxazolyl, phenyl, pyridyl, pyrryl, quinolyl, quinoxalyl, tetrazoyl, thiazoyl, thienyl and the like. Substituted aryl refers to an aryl group substituted by, for example, one to four substituents such as substituted and unsubstituted alkyl, alkenyl, alkynyl, and aryl; halo; trifluoromethoxy; trifluoromethyl; hydroxy; alkoxy; cycloalkyloxy; heterocyclooxy; alkanoyl; alkanoyloxy; amino; alkylamino; aralkylamino; cycloalkylamino; heterocycloamino; dialkylamino; alkanoylamino; thio; alkylthio; cycloalkylthio; heterocyclothio; ureido; nitro; cyano; carboxy; carboxyalkyl; carbamyl; alkoxycarbonyl; alkylthiono; arylthiono; alkylsulfonyl; sulfonamido; aryloxy; and the like. The substituent may be further substituted, for example, by halo, hydroxy; alkyl, alkoxy; aryl, substituted aryl, substituted alkyl, substituted aralkyl, and the like.
The terms xe2x80x9calkylarylxe2x80x9d or xe2x80x9carylalkylxe2x80x9d refer to an aryl group bonded directly through an alkyl group, such as benzyl. Similarly, xe2x80x9calkenylarylxe2x80x9d and xe2x80x9carylalkenylxe2x80x9d refer to an aryl group bonded directly through an alkenyl group and xe2x80x9calkynylarylxe2x80x9d and xe2x80x9carylalkynylxe2x80x9d refer to an aryl group bonded directly through an alkynyl group.
The term amidoalkylaryl refer to a group of the formula xe2x80x94ZNHxe2x80x94(Cxe2x95x90O)xe2x80x94Rxe2x80x2-Rxe2x80x3 where Z may be present or absent, and Z and Rxe2x80x2 are each independently an optionally substituted C1-C10 alkyl, alkenyl, or alkynyl and Rxe2x80x3 is an optionally substituted aryl.
The terms xe2x80x9chalogen,xe2x80x9d xe2x80x9chaloxe2x80x9d, or xe2x80x9chalidexe2x80x9d refer to fluorine, chlorine, bromine and iodine.
The term xe2x80x9cerythromycinxe2x80x9d refers to a compound of the formula 
where R, R1, R2, R3, R4, R5, R6, and R8 are as described herein and derivatives and analogs thereof.
Free hydroxyl groups in the compounds of the present invention may optionally be protected with a hydroxyl protecting group. The term xe2x80x9chydroxy protecting groupxe2x80x9d refers to groups known in the art for such purpose. Commonly used hydroxy protecting groups are disclosed, for example, in T. H. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 2nd edition, John Wiley and Sons, New York (1991), which is incorporated herein by reference. Illustrative hydroxyl protecting groups include but not limited to tetrahydropyranyl; benzyl; methylthiomethyl; ethythiomethyl; pivaloyl; phenylsulfonyl; triphenylmethyl; trisubstituted silyl such as trimethyl silyl, triethylsilyl, tributylsilyl, tri-isopropylsilyl, t-butyldimethylsilyl, tri-t-butylsilyl, methyldiphenylsilyl, ethyldiphenylsilyl, t-butyldiphenylsilyl and the like; acyl and aroyl such as acetyl, pivaloylbenzoyl, 4-methoxybenzoyl, 4-nitrobenzoyl and aliphatic acylaryl and the like. Hydroxyl protected versions of the inventive compounds are also encompassed within the scope of the present invention.
In addition to the explicit substitutions at the above-described groups, the inventive compounds may include other substitutions where applicable. For example, the erythromycin backbone or backbone substituents may be additionally substituted (e.g., by replacing one of the hydrogens or by derivatizing a non-hydrogen group) with one or more substituents such as C1-C5 alkyl, C1-C5 alkoxy, phenyl, or a functional group. Illustrative examples of suitable functional groups include but are not limited to alcohol, sulfonic acid, phosphine, phosphonate, phosphonic acid, thiol, ketone, aldehyde, ester, ether, amine, quaternary ammonium, imine, amide, imide, imido, nitro, carboxylic acid, disulfide, carbonate, isocyanate, carbodiimide, carboalkoxy, carbamate, acetal, ketal, boronate, cyanohydrin, hydrazone, oxime, hydrazide, enamine, sulfone, sulfide, sulfenyl, and halogen.
The term xe2x80x9csubjectxe2x80x9d as used herein, refers to an animal, preferably a mammal, most preferably a human, who has been the object of treatment, observation or experiment.
The term xe2x80x9ctherapeutically effective amountxe2x80x9d as used herein, means that amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician, which includes alleviation of the symptoms of the disease or disorder being treated.
As used herein, the term xe2x80x9ccompositionxe2x80x9d is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combinations of the specified ingredients in the specified amounts.
Compounds of the Present Invention
In one embodiment, the present invention provides compounds of the formulas 
wherein:
R is hydroxyl or methoxy;
R1 is selected from the group consisting of hydrogen, hydroxyl, halide, NH2, OR9, 
where R9 is C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, aryl or heteroaryl and R10 and R11 are each independently hydrogen, C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, or aryl;
R2 and R3 are each independently selected from the group consisting of hydrogen, C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, aryl, alkylaryl, alkenylaryl, alkynylaryl or R2 and R3 together form a cycloalkyl or a cycloaryl moiety;
R4 is hydrogen or methyl;
R5 is hydrogen, hydroxyl, oxo, or together with R6 and the carbons to which they are attached form a cyclic carbonate;
R6 is hydrogen, hydroxyl, OR12 where R12 is C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, or together with R5 and the carbons to which they are attached form a cyclic carbonate;
R7 is methyl, C3-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, alkylaryl, alkenylaryl, alkynylaryl, amidoalkylaryl, amidoalkenylaryl, or amidoalkynylaryl;
R8 is C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, alkylaryl, alkenylaryl, alkynylaryl, amidoalkylaryl, amidoalkenylaryl, or amidoalkynylaryl; and,
x is a single or a double bond.
In another embodiment, the present invention provides compounds of the formulas 
wherein R, R1, R2, R3, R4, R6, R7, R8 and x are as described previously.
In another embodiment, the present invention provides compounds of the formulas I, II, III, and IV wherein: R is hydroxyl or methoxy; R1 is hydrogen, hydroxyl, fluoro; R2 and R3 are each independently C1-C5 alkyl, phenyl or benzyl; R4 is methyl; R5 is hydrogen, hydroxyl or oxo; R6 is hydrogen, hydroxyl, or C1-C5 alkoxy; R7 is methyl, C3-C5 alkyl, C2-C5 alkenyl, C2-C5 alkynyl, aryl, alkylaryl or alkenylaryl; R8 is C1-C5 alkyl, C2-C5 alkenyl, C2-C5 alkynyl, aryl, alkylaryl or alkenylaryl; and, x is single bond or a double bond.
In another embodiment, the present invention provides compounds of the formulas I, II, III, and IV wherein: R is hydroxyl or methoxy; R1 is hydrogen or hydroxyl; R2 is methyl; R3 is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, secbutyl, or tertbutyl; R4 is methyl; R5 is hydrogen, hydroxyl or oxo; R6 is hydrogen, hydroxyl, or methoxy; R7 is methyl, vinyl, propyl, isobutyl, pentyl, prop-2-enyl, propargyl, but-3-enyl, 2-azidoethyl, 2-fluoroethyl, 2-chloroethyl, cyclohexyl, phenyl, or benzyl; R8 is methyl, ethyl vinyl, propyl, isobutyl, pentyl, prop-2-enyl, propargyl, but-3-enyl, 2-azidoethyl, 2-fluoroethyl, 2-chloroethyl, cyclohexyl, phenyl, or benzyl; and, x is a single or a double bond.
In another embodiment, the present invention provides compounds of the formulas I, II, III, and IV wherein: R is methoxy; R1 is hydrogen or hydroxyl; R2 is methyl; R3 is methyl, ethyl, or isopropyl; R4 is methyl; R5 is hydrogen, hydroxyl or oxo; R6 is hydrogen, hydroxyl, or methoxy; R7 is propyl, but-3-enyl, 2-azidoethyl, phenyl, or benzyl; R8 is ethyl, propyl, but-3-enyl, 2-azidoethyl, phenyl, or benzyl; and, x is a single or a double bond.
In another embodiment, the present invention provides compounds of the following formulas 
wherein R1 is hydrogen or hydroxyl; R3 is methyl, ethyl, or isopropyl; R6 is hydrogen, hydroxyl, or methoxy; R7 is propyl; and R9 is ethyl or propyl.
In another embodiment, the present invention provides compounds of the following formulas 
wherein R1 is hydrogen or hydroxyl; R3 is methyl, ethyl, or isopropyl; R6 is hydrogen, hydroxyl; R7 is propyl; and R8 is ethyl or propyl.
Starting Materials
The compounds of the present invention can be prepared in accordance with the methods of the present invention by a combination of recombinant DNA technology and organic chemistry.
Recombinant techniques are used to provide, in many instances, xe2x80x9cunnaturalxe2x80x9d erythromycins or erythromycin derivatives that differ in one or more positions from the naturally occurring erythromycins A, B, C, or D. Although any suitable recombinant means may be used, a useful starting point is the complete 6-dEB synthase gene cluster that has been cloned in vectors and thus is amenable to genetic manipulations in E. coli and expression of the polyketide in Streptomyces. See U.S. Pat. Nos. 5,672,491; 5,830,750; 5,843,718; 5,712,146; and 5,962,290 which are all incorporated herein by reference. Once the aglycone is formed, it is next hydroxylated and/or glycoslyated and/or methylated at the appropriate positions by a converter strain that possesses the desired functionalities.
A particularly useful converter strain is an Saccharopolyspora erythraea eryA mutant that is unable to produce 6-dEB but can still carry out the desired conversions (Weber et al., J. Bacteriol. 164(1): 425-433 (1985). This mutant strain is able to take exogenously supplied 6-dEB and process it to erythromycin A by converting it into erythronolide B, 3-xcex1-mycarosylerythronolide B, erythromycin D, erythromycin C, and finally to erythromycin A. An alternative route to erythromycin A is through erythromycin B where exogenously supplied 6-dEB is converted into erythronolide B, 3-xcex1-mycarosylerythronolide B, erythromycin D, erythromycin B, and finally to erythromycin A. Other mutant strain, such as eryB, eryC, eryG, and/or eryK mutants, or mutant strains having mutations in multiple genes can be used to make compounds having any combinations of hydroxylations at C-6 and C-12, glycosylations at C-3 and C-5, and methylation at C-3xe2x80x3xe2x80x94OH. Any of these products may be used as starting materials for the practice of the present invention.
Erythromycins where the substituent at C-13 is methyl or ethyl, the 6-deoxyerythronolide B synthase (xe2x80x9cDEBSxe2x80x9d) from S. erythraea can be used in a recombinant expression system described in U.S. Pat. No. 5,672,491 to produce the aglycone in Streptomyces coelicolor. Optionally, the oleandolide or megalomicin polyketide synthase (xe2x80x9cPKSxe2x80x9d) genes may be used in this expression system. See U.S. Provisional Patent Application Serial No. 60/158,305 filed Oct. 8, 1999 and utility application Ser. No. 09/679,279, filed Oct. 4, 2000, entitled Recombinant Megalomicin Biosynthetic Genes by inventors Robert McDaniel and Yana Volchegursky; and PCT Publication No. WO 00/026,349 which are all incorporated herein by reference.
For erythromycins where the substituent at C-13 is something other than methyl or ethyl, one can employ a technique known as chemobiosynthesis in which activated thioesters called SNAC-diketides are converted to 13-substituted 6-dEB derivatives (13-R-13-desethyl-6-dEB compounds) by fermentation of S. coelicolor CH999/pJRJ2 or functionally similar strains that contain a PKS in which the ketosynthase domain of module 1 has been inactivated by mutation (the KS1xc2x0 mutation). This methodology is described in PCT Publication Nos. WO 97/02358 and WO 99/03986 and U.S. Pat. No. 6,066,721 which are all incorporated herein by reference. Additional SNAC-diketide compounds and the corresponding aglycones are described in PCT Publication No. WO 00/44717 which is incorporated herein by reference.
6-dEB and 6-dEB derivatives such as 13-substituted 6-dEB are converted into the desired erythromycin starting material by an appropriate converter strain. For example, any one of the post PKS products may be used as starting materials such as 13-substituted counterparts (where the ethyl group which normally exists at C-13 is replaced with another substituent) to: erythronolide B, 3-xcex1-mycarosylerythronolide B, erythromycin D, erythromycin B, erythromycin C, and erythromycin A. In particular, 13-substituted erythromycin A can be made by fermentation with an eryA mutant that is incapable of producing 6-dEB but can still carry out the desired conversions. 13-substituted erythromycin B can be made by fermentation with an eryA mutant that is incapable of producing 6-dEB and in which the ery K (12-hydroxylase) gene has been deleted or otherwise rendered inactive. Alternatively, erythromycin B derivatives can be made in a KS1xc2x0/eryK mutant strain of S. erythaea. The general method for using chemobiosynthesis for making modified 6-dEB is illustrated by Example 1 with specific reference to 13-propyl-6-dEB (13-propyl-13-desethyl-6-dEB). The general method for converting modified 6-dEB compounds to the desired hydroxylated and glycosylated form by using an eryA converter strain is illustrated by Example 2 with specific reference to converting 13-propyl 6-dEB to 13-propyl erythromycin A (13-propyl-13-desethyl-erythromycin A).
6-Desmethyl erythromycins, a starting material for making the furanyl erythromycins (compounds of formula II or IV) of the present invention, are made by replacing the acyl transferase (xe2x80x9cATxe2x80x9d) domain of module 4 (encoding a 6-methyl group) of a 6-dEB or 8,8a-deoxyoleandolide synthase with an AT a malonyl specific AT domain (encoding a 6-hydrogen) to provide the 6-desmethyl analog of the erythromycin aglycone. Illustrative examples of malonyl specific AT domains include AT2 and AT12 of rapamycin; AT3 and AT4 of epothilone; and AT10 of FK-520.
Alternatively, the AT4 domain of 6-dEB or 8,8a-deoxyoleandolide polyketide synthase is mutated to correspond to AT domains more characteristic of AT domains having malonyl specificity. More particularly, three mutations are made. In the first, nucleotides 6214-6227 of the open reading frame encoding AT4 (CGC GTC GAC GTG CTC) is modified to the sequence, GAC GAC CTC TAC GCC where bold indicates the altered nucleotide, to change the encoded amino acids from RVDVLQ to DDLYA. In the second, nucleotides 6316-6318 (CAG) is modified to the sequence CTC to change the encoded amino acid from Q to L. In the third, nucleotides 6613-6621 (TAC GCC TCC) is modified to the sequence CAC GCC TTC to change the encoded amino acids from YAS to HAF.
In either case, the resulting aglycone is bioconverted to 6-desmethyl erythromycin as described above although some modification for C-6 hydroxylation may be required. For example, the specificity of the native eryF gene product may need to be altered to accept the 6-desmethyl substrate or the use of a different P450 oxidase may be required.
Other starting materials include 6-hydroxy-erythromycin (where the methyl at C-6 has been replaced with a hydroxyl group), 6-oxo erythromycin (where the methyl at C-6 has been replaced with an oxo group), 6-methoxy erythromycin (where the methyl at C-6 has been replaced with a methoxy group) and 6-desmethyl, 7-hydroxy-erythromycin. In one embodiment, 6-OH, 6-OMe erythromcyins are made by replacing AT4 of 6-dEB or 8,8a-deoxyoleandolide synthase with an AT domain encoding hydroxymalonate or methoxymalonate. See PCT Publication WO 00/20601 which is incorporated herein by reference. The 6-OH and 6-OMe aglycone is bioconverted to 6-desmethyl-6-hydroxy erythromycin and 6-desmethyl-6-methoxy erythromycin respectively by fermentation with an appropriate eryA mutant that is incapable of producing 6-dEB and in which the eryF (C-6 hydroxylase) function has been deleted or otherwise inactivated. Fermentation of 6-OH or 6-OMe aglycone with an eryA mutant that possesses eryF (or equivalent) function lead to the 6-desmethyl-6-oxo erythromycin.
In one embodiment, 6-desmethyl, 7-hydroxy erythromycins are made by replacing AT4 of a 6-dEB or 8,8a-deoxyoleandolide polyketide synthase with a malonyl specific AT as described above as well as deleting or otherwise inactivating the dehydratase activity of module 3 (xe2x80x9cDH3xe2x80x9d). The resulting 6-desmethyl, 7-hydroxy aglycone is converted into the corresponding erythromycin derivative by fermentation with an appropriate eryA mutant that is incapable of producing 6-dEB as described above.
Synthetic Methods
The methods described herein are generally applicable to all erythromycins and erythromycin derivatives (e.g., erythromycins A, B, C, and D, 13-substituted erythromycins A, B, C, and, D, erythronolide B, 3-xcex1-mycarosylerythronolide B, and derivatives thereof) unless explicitly limited. As such, references to specific embodiments are for the purposes of illustration only and are not intended to limit in any way the scope of the present invention.
In one aspect of the present invention, methods for forming the 8,9-anyhydro erythromycin 6,9-enol ether and 6,9 epoxide compounds are provided. The 8,9-anhydro erythromycin 6,9-enol ethers are also referred to as enol ethers or dihydrofurans. The 6,9-epoxides are also referred to as epoxides or tetrahydrofurans. Scheme 1A illustrates one embodiment for making the enol ether and epoxide compounds from erythromycin A and B derivatives (where R6 is hydrogen or hydroxyl and R7 is as previously described). 
In general, the enol ether compounds 11 are formed by treating with mild acid the desired erythromycin starting material such as 10 (see Example 3). The corresponding epoxide 12 is formed by reducing the carbon-carbon double bond between C-8 and C-9 of the enol ether 11 (see Example 4). Scheme 1B illustrates another embodiment for making epoxide 12 also from compound 10. 
The free hydroxyls of erythromycin 10 are protected and the C-9 ketone is reduced with sodium borohydride to a 9-dihydro erythromycin intermediate (where C-9 is xe2x80x94CHOHxe2x80x94). The hydroxyl group at C-9 is subsequently activated and displaced. The protecting groups are then removed to yield compound 12. Examples 12-17 describe this protocol in greater detail starting from erythromycins A and B (which are specific embodiments of compound 10) and their 4xe2x80x3-deoxy counterparts as starting material.
In another aspect of the present invention, methods for demethylating one or both of the 3xe2x80x2-N-methyl groups are provided. The demethylated 3xe2x80x2-nitrogen then may be subsequently reacted with an alkyl or an aryl group. The 3xe2x80x2-N demethylation and subsequent alkylation (or arylation) may be performed using erythromycins, enol ethers, or epoxide derivatives. Because these methods can be used on a wide variety of starting materials, the timing of these reactions is determined by the desired modifications at other macrolide positions. Scheme 2 illustrates one embodiment where the demethylation and alkylation reactions occur after the enol ether formation. 
Enol ether 11, formed from erythromycin 10 as described previously by Scheme 1, is demethylated at the 3xe2x80x2-N by treatment with light, iodine and sodium acetate. Additional reagents and longer reaction times will remove both methyl groups if desired. The demethylated enol ether 12 is then alkylated or arylated with the appropriate alkyl halide or aryl halide to yield compound 13. Enol ether 13 may be optionally reduced to form its 6,9 epoxide counterpart using the procedures described by Scheme 1A. Examples 6 and 7 illustrate the demethylation and subsequent alkylation protocol with respect to erythromycin 10.
In another aspect of the present invention, methods for forming 11-oxo compounds are provided. Scheme 3 illustrates one embodiment with respect to 11-oxo-erythromycin A derivatives. 
Enol ether 14, a 2xe2x80x2 and 4xe2x80x3 protected form of compound 13, is oxidized (e.g., using a carbodiimide and methylsulfoxide or a hypervalent iodine species) to yield compound 15. Deprotection at the 2xe2x80x2 and 4xe2x80x3 positions yields the unprotected form of compound 15. These protocols are described in greater detail in Examples 8-9. Alternatively, the hydroxyl at the C-12 position of compound 15 may be optionally alkylated to yield compound 16 after deprotection (see Example 10).
In another aspect of the present invention, methods for forming 11-hydrogen compounds are provided. Scheme 4 illustrates one embodiment for making 11 hydrogen counterparts from erythromycin B derivatives. 
Compound 14a, the erythromycin B counterpart to compound 14 described in Scheme 3, is deoxygenated as described by Scheme 4 to yield compound 17. Deoxygenation of the 11-O-xanthate is illustrated, although other substrates such as the thiocarbonylimidazolide are also suitable intermediates. Compound 17 may be deprotected at the 2xe2x80x2 and 4xe2x80x3 positions to yield the unprotected form. Alternatively, as shown by Scheme 4, compound 17 may be reduced to yield the corresponding 6,9 epoxide 18 after deprotection.
In another aspect of the present invention, methods for making 4xe2x80x3-desoxy compounds are provided. Scheme 5 illustrates one embodiment. 
Erythromycin 10 is acetylated at the 2xe2x80x2 hydroxyl to yield compound 19 (Example 12). The 2xe2x80x2-O-acetyl erythromycin 19 is then treated with thiocarbonyldiimidazole and 4-dimethylaminopyridine in dichloromethane. The resulting product is isolated and treated with tributyltin hydride to yield compound 20 (see Example 13). The 4xe2x80x3-desoxy erythromycin may be used in any combination of the protocols described by Schemes 1-4 to make the corresponding 4xe2x80x3-desoxy counterparts. Alternatively, the 4xe2x80x3-hydroxyl of erythromycin 10 may be modified to other groups (e.g., halide, NH2, alkoxy and aryloxy) using standard chemical reactions that are known in the art and used similarly as starting materials for the protocols described herein. See e.g. Advanced Organic Chemistry 3rd Ed. by Jerry March (1985) which is incorporated herein by reference.
In another aspect of the present invention, methods for forming furanyl erythromycins are provided. In one embodiment, furanyl erythromycins are prepared synthetically by demethylating the naturally occurring methyl group at C-6. For example, a suitably protected erythromycin is converted to the 6-O-xanthate via reaction with carbon disulfide and methyl iodide, and the xanthate is pyrolyzed to yield 6,6a-anhydroerythromycin. Ozonolysis yields the 6-oxo-erythromycin, which can be converted to the 6,9-epoxide by dehydration from treatment with mild acid or acetic anhydride. Alternatively, the 6-oxo-erythromycin may be prepared recombinantly as described previously. Scheme 6 illustrates another embodiment using 6-desmethyl erythromycins. 
6-Desmethyl erythromycin 21 (where R1 and R6 hydrogen or hydroxyl and R8 is as previously described) is treated with mild acid such as dichloroacetic acid to form enol ether 22. Compound 22 is then treated with a mild oxidizing agent such as bromine in base to yield furanyl erythromycin 23. In yet another embodiment, 7-hydroxy-8,9-anhydro erythromycin 6,9-hemiacetal (22 where Rxe2x80x2 is hydroxyl, and R6 is hydrogen or hydroxyl and R8 is as previously described) is mesylated and subjected to base-catalyzed elimination to yield furanyl erythromycin 23.
In another aspect of the present invention, methods for forming 12-membered enol ethers are provided. Scheme 6 illustrates one embodiment for making these compounds. 
Erythromycin enol ether 11 is treated with potassium carbonate in methanol to yield the 12 membered enol ether 24 (see Example 5). A 12 membered 6,9 epoxide may be made using the same procedure by starting with the 6,9 epoxide form of erythromycin such as compound 12 instead of compound 11. A 12 membered furanyl compound can be made using the same procedure by starting with furanyl erythromycin 23 instead of compound 11.
Methods of Use
In general, methods of using the compounds of the present invention comprise administering to a subject in need thereof a therapeutically effective amount of a compound of the present invention. Illustrative examples of disorders that may be treated with the inventive compounds include but are not limited to gastroparesis, gastroesophageal reflux disease, anorexia, gall bladder stasis, postoperative paralytic ileus, scleroderma, intestinal pseudoobstruction, gastritis, emesis, and chronic constipation (colonic inertia).
The therapeutically effective amount can be expressed as a total daily dose of the compound or compounds of this invention and may be administered to a subject in a single or in divided doses. The total daily dose can be in amounts, for example, of from about 0.01 to about 25 mg/kg body weight, or more usually, from about 0.1 to about 15 mg/kg body weight. Single dose compositions may contain such amounts or submultiples thereof as to make up the daily dose. In general, treatment regimens according to the present invention comprise administration to a subject in need of such treatment of from about 10 mg to about 1000 mg of the compound(s) of the present invention per day in single or multiple doses.
Typically, the inventive compound will be part of a pharmaceutical composition or preparation which may be in any suitable form such as solid, semisolid, or liquid form. In general, the pharmaceutical preparation will contain one or more of the compounds of the invention as an active ingredient and a pharmaceutically acceptable carrier. Typically the active ingredient is in admixture with an organic or inorganic carrier or excipient suitable for external, enteral, or parenteral application. The active ingredient may be compounded, for example, with the usual non-toxic, pharmaceutically acceptable carriers for tablets, pellets, capsules, suppositories, pessaries, solutions, emulsions, suspensions, and any other form suitable for use. Oral dosage forms may be prepared essentially as described by Hondo et al., 1987, Transplantation Proceedings XIX, Supp. 6: 17-22, incorporated herein by reference.
The carriers that can be used include water, glucose, lactose, gum acacia, gelatin, mannitol, starch paste, magnesium trisilicate, talc, corn starch, keratin, colloidal silica, potato starch, urea, and other carriers suitable for use in manufacturing preparations, in solid, semi-solid, or liquified form. In addition, auxiliary stabilizing, thickening, and coloring agents and perfumes may be used. For example, the compounds of the invention may be utilized with hydroxypropyl methylcellulose essentially as described in U.S. Pat. No. 4,916,138, incorporated herein by reference, or with a surfactant essentially as described in EPO patent publication No. 428,169, incorporated herein by reference.
In summary, the present invention provides novel macrolide compounds, methods for making and methods of using the same which are further illustrated by the following examples.